Methods of reducing extravasation of inflammatory cells

ABSTRACT

A method for modifying access of cells to extravascular spaces and regions comprising administering to a patient an enzyme that cleaves chondroitin sulfate proteoglycans is provided. It has been found that administration of an enzyme that cleaves chondroitin sulfate proteoglycans to a patient disrupts extravasation of cells from the blood stream into tissue. The present invention provides methods of reducing penetration of cells associated with inflammation into tissue of a patient. Several methods are also provided for the regulation and suppression of inflammation comprising administering enzymes that digest chondroitin sulfates. Also provided are methods of treating and preventing inflammation associated with infection, injury and disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of pending U.S. applicationSer. No. 10/847,636, filed May 17, 2004, which claims priority to U.S.Provisional Application No. 60/471,189, filed May 16, 2003, each ofwhich are herein incorporated by reference in their entirety.

BACKGROUND

1. Field of the Invention

The present disclosure relates to methods of reducing penetration ofinflammatory cells into tissue, in particular to methods of reducingextravasation of macrophage cells from blood vessels, methods ofpreventing, regulating and suppressing inflammatory response, andmethods of treating inflammatory states.

2. Description of Related Art

The inflammatory response evolved to protect organisms against injuryand infection. Following an injury or infection, a complex cascade ofevents leads to the delivery of blood-borne leukocytes to sites ofinjury to kill potential pathogens and promote tissue repair. However,the powerful inflammatory response has the capacity to cause damage tonormal tissue, and dysregulation of the innate or acquired immuneresponse is involved in different pathologies. It has long been knownthat Multiple Sclerosis is an inflammatory disease of the brain, but itis only in recent years that it has been suggested that inflammation maysignificantly contribute to diseases such as stroke, traumatic braininjury, HIV-related dementia, Alzheimer's disease and prion disease. Therecognition of an inflammatory component in the pathology of these andother diseases has come from the development of new techniques andreagents for the study of inflammation biology.

The inflammatory response is a part of innate immunity. Inflammationoccurs when tissues are injured by viruses, bacteria, trauma, chemicals,heat, cold, or any other harmful stimuli. Chemicals, such as bradykinin,histamine, and serotonin, are released by specialized cells and attracttissue macrophages and white blood cells to localize in an area toengulf (phagocytize) and destroy foreign substances. A byproduct of thisactivity is the formation of pus—a combination of white blood cells,bacteria, and foreign debris. The chemical mediators released during theinflammatory response give rise to the typical findings associated withinflammation.

Inflammation constitutes the body's response to injury and ischaracterized by a series of events that includes the inflammatoryreaction per se, a sensory response perceived as pain, and a repairprocess. The inflammatory reaction is characterized by successivephases: (1) a silent phase, where cells, including resident cells in thedamaged tissue, release the first inflammatory mediators, (2) a vascularphase where vasodilation and increased vascular permeability occur, and(3) a cellular phase, which is characterized by the infiltration ofleukocytes to the site of injury. The repair process includes tissuecell division, neovascularization and reinnervation of repaired tissues.In many diseases such as arthritis, inflammatory bowel disease, andasthma, the inflammatory process is not appropriately regulated. As aresult, significant tissue dysfunction (leading to the generation of thesymptoms that typify these diseases), and tissue re-structuring occur(e.g., fibrosis) that can further impair tissue function.

The very first event of the inflammatory reaction, the “silent phase” isbased upon the reaction of cells and resident cells of the damagedtissue. These resident cells release mediators, such as nitric oxide(NO), histamine, kinins, cytokines, or prostaglandins. The release ofthese vasomotor mediators from resident cells leads to the second phaseof the inflammatory reaction: the vascular phase. Vascular tone andpermeability are regulated by an endothelial-dependent mechanisminvolving the release of nitric oxide. Certain agonist signal to sensoryafferents, including the release of neuropeptides which are known to acton vascular beds to induce vasodilation and increased permeability.Increased vasodilatation and permeability provoke plasma leakage fromthe blood to the inflamed tissues, and facilitate the passage ofleukocytes from the blood flow to the tissues.

The third phase of the inflammatory process is the cellular phase, whichis characterized by the arrival of leukocytes circulating in the blood.In order to be recruited to the site of inflammation, circulatingleukocytes roll onto the venular endothelial surfaces, adhere to theendothelium, and then transmigrate across the endothelial barrier. Theprocess by which cells leave the blood stream and penetrate tissueparenchyma is known as extravasation. The events of rolling, adhesion,and transmigration (extravasation) are regulated by several celladhesion molecules, known as CAMs and receptor molecules expressed bythe endothelium and the leukocytes.

The inflammatory response to tissue damage may be of great value. Byisolating the damaged area, mobilizing effector cells and molecules tothe site, and—in the late stages—promoting healing, inflammation mayprotect the body. However, inflammation is more often associated withpain, injection and diseased states. Often the inflammatory response isout of proportion to stimulus which activated the response. Theinflammatory process inevitably causes tissue damage and is accompaniedby simultaneous attempts at healing and repair. Tissue destruction iscaused by both the caustic agents and by the inflammatory processitself. The result can be more damage to the body than the agent itselfwould have produced. For example, all the many types of allergies andmany of the autoimmune diseases are examples of inflammation in responseto what should have been a harmless, or at least a noninfectious, agent.Some examples of chronic inflammatory diseases include Asthma,Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), Systemic LupusErythematosus (SLE), psoriasis, and Chronic Obstructive PulmonaryDisease (COPD). In many of these cases, the problem is made worse by theformation of antibodies against self antigens or persistent antigensfrom smoldering infections. Additionally, any disease with aninflammatory component may be treated by a better understanding of theimmune system and the disease-fighting responses to toxins, injury,viruses and bacteria. Recently, it has been shown that inflammation hasbeen link to cardiovascular diseases.

Traditional therapeutics for the treatment of inflammation includeanti-inflammatory agents and steroids. Inappropriate inflammation can betreated with steroids like the glucocorticoid cortisol, nonsteroidalanti-inflammatory drugs (NSAIDs) like aspirin and ibuprofen, and anumber of proteins produced by recombinant DNA technology.

The NSAIDs achieve their effects by blocking the activity ofcyclooxygenase. The body produces several different forms ofcyclooxygenase (COX), including COX-1, which is involved in pain,clotting, and protecting the stomach; COX-2, which is involved in thepain produced by inflammation. Most of the NSAIDs inhibit both COX-1 andCOX-2. However, some newer drugs, the so-called COX-2 inhibitors, suchas rofecoxib (Vioxx®) and celecoxib (Celebrex®) are much more activeagainst COX-2 than COX-1.

Recombinant DNA and monoclonal antibody technology have produced somenew therapies that are being enlisted in the battle against damaginginflammation. Examples of these therapeutics include: (1) an IL-1antagonist that binds and inactivates the IL-1 receptor; (2) etanercept(Embrel®), which a soluble version of the TNF-α receptor, which bindsTNF-α preventing it from carrying out its many inflammatory actions; (3)recombinant protein C, which helps the body dissolve the tiny clots thatare triggered during inflammation; (4) infliximab (Remicade®), which isa monoclonal antibody that binds to TNF-α, particularly promisingagainst some inflammatory diseases such as rheumatoid arthritis; and (5)the antibody natalizumab (Antegren®; Biogen Inc., Cambridge Mass.),which functions by blocking the adhesion of immune cells to bloodvessels and can inhibit movement of immune cells from the blood into thebrain. Several of these therapies carry a severe risk of allowinginfections to develop. In fact, the more powerful the anti-inflammatoryagents (e.g., glucocorticoids), the greater the risk of infection.

Reducing inflammation and regulating the inflammatory response also isbeneficial in the prevention of various cancers and other cellconditions. Chronic inflammation is a recognized cause of cancer. Forexample, liver cancer is often the sequel to years of inflammationcaused by infection hepatitis B or C infection. Lung cancer often is theend stage of years of chronic inflammation caused by inhaled irritants,such as tobacco smoke. Cervical cancer can follow chronic infection andinflammation caused by papilloma viruses and chlamydia. Similarly,bladder, colon, pancreas, stomach, and other cancers may be the finalstage of years of inflammation.

The strong link between chronic inflammation and cancer should not besurprising considering that the reactive oxygen species (ROS) liberatedduring inflammation are powerful DNA-damaging agents. Additionally,increased mitosis in response to inflammation puts more cells at risk ofmutations as they replicate their DNA during S phase. Furthermore,apoptosis, the programmed death of damaged cells, is suppressed ininflamed tissue. So precancerous cells with genetic mutations, whichshould have committed suicide, continue and ultimately develop intocancerous cells. Therefore, the regulation of the inflammatory responseincluding reducing inflammation in tissues is beneficial to theprevention of various cancers.

The penetration of inflammatory cells into and through the parenchyma oftissues is intimately involved in the pathophysiology of numerousdisease, injury and inflammatory states. The ability to preventpenetration of inflammatory cells into tissue has beneficial therapeuticeffects. It is known that reduction of tissue penetrating inflammatorycells has been accomplished to a degree with steroid, anti-integrin andother anti-inflammatory compounds. The access of cells through the bloodvessel wall and into tissues is, in part, dependent upon theextracellular matrix, receptor molecules, CAMs, the cell membranes ofthe vascular endothelium and the migrating cell. Interfering with one ormore of these signals or receptors disrupts the recruitment ofinflammatory cells to the tissue. This disruption thus leads to reducedinflammation in the tissue.

SUMMARY OF THE INVENTION

It has been found that the use of enzymes capable of cleavingchondroitin sulfate proteoglycans are effective in altering theextravasation of cells associated with inflammation, such as macrophagesfrom blood vessels into tissue.

One embodiment of the present invention is a method for modifying accessof cells to extravascular spaces and regions comprising administering toa patient an enzyme that cleaves chondroitin sulfate proteoglycans.Another embodiment of the present invention is a method of reducingpenetration of cells associated with inflammation into tissue of apatient. In several methods of the invention, an enzyme selected fromthe group consisting of chondroitinase ABC_(Type I), chondroitinaseABC_(Type II), chondroitinase AC, chondroitinase B, Hyaluronidase 1,Hyaluronidase 2, Hyaluronidase 3, Hyaluronidase 4, andhyaluronoglucosaminidase, fragments thereof, and combinations thereofare used. In several embodiments of the invention enzymes directedagainst integrins containing chondroitin sulfate proteoglycan expressedon the cell surface of cells are used.

Another embodiment of the present invention is a method for inhibitingextravasation of cells associated with inflammation from blood vesselscomprising administering to a patient an enzyme that cleaves chondroitinsulfate proteoglycans. In several embodiments of the invention an enzymeprevents cells selected from the group selected from the groupconsisting of white blood cells, leukocytes, neutrophils, eosinophils,basophils, lymphocytes, B-cells, T-cells, monocytes, and macrophagescells from leaving the blood stream.

Another embodiment of the invention is a method of treating inflammationin a patient comprising administering to the patient an enzyme thatcleaves chondroitin sulfate proteoglycans. In several embodiments of thepresent invention, inflammation is associated with disease or injury,such as chronic inflammatory diseases and central nervous systemdisease.

Another embodiment of the invention is a method of preventinginflammation in a patient comprising administering to the patient anenzyme that cleaves chondroitin sulfate proteoglycans.

Another embodiment of the present invention is a method of treatinginflammation of a patient comprising extracting cells associated withinflammation from a patient, subjecting the cells to an enzyme thatcleaves chondroitin sulfate proteoglycans ex vivo to modify the cells,and administering the modified blood cells into the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings, in which:

FIG. 1 is schematic drawing of the four major families of cell adhesionmolecules.

FIG. 2 is a schematic drawing of the sequence of cell-cell interactionsleading to tight binding of leukocytes to activated endothelial cell andsubsequent extravasation.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toan “enzyme” is a reference to one or more enzymes and equivalentsthereof known to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It has been found that the administration of certain enzymes to theblood stream disrupts the mechanism by which cells move from the bloodstream to the tissues. When cells, primarily macrophages and otherimmunological cells or cells associated with inflammation, are signaledto enter a tissue, extravasation ensues. The cells circulating in theblood stream migrate to the blood vessel walls where they encounterspecific receptors, known as cell adhesion molecules. Through specificreceptor based adhesion to the vessel wall, the cells roll along thewall until they encounter an endothelial cell junction where they cansqueeze out of the vessel and enter the extravascular space. Some ofthese adhesion molecules or receptors contain carbohydrate chains madeup of chondroitin sulfates. Digestion of these chains with enzymesinterrupts extravasation. Several methods are provided for theregulation, prevention, treatment and suppression of the inflammatoryresponse comprising administering enzymes that digest chondroitinsulfates. Also provided are methods of treating and preventing infectionand diseases associated with inflammatory response.

The process of “extravasation” is known as the transmigration of cells,such as leukocytes, from a blood vessel into the extravascular space,and may further include migration into surrounding tissue. As usedherein the term “leukocyte” is used to refer to the class of cellsassociated with inflammation, which may also be defined as any of thevarious blood cells that have a nucleus and cytoplasm. Also known aswhite blood cells, leukocytes include neutrophils, eosinophils,basophils, lymphocytes, such as B-cells, T-cells, monocytes andmacrophages. Four types of leukocytes are particularly important inimmune defense, including neutrophils, which release severalantibacterial proteins; monocytes, which are the precursors ofmacrophages that engulf and destroy foreign particles, and T and Blymphocytes, which are the antigen-recognizing cells of the immunecells.

The term “therapeutic agent” used in connection with the inflammatoryresponse therefore means an agent that functions as a blocker,suppressor, regulator or modulator of the inflammatory response. Forexample, several therapeutic enzymes described herein function asblockers, suppressors, regulators or modulators of the inflammatoryresponse.

Inflammation is a defense reaction caused by tissue damage or injury,characterized by redness, heat, swelling, and pain. The inflammatoryresponse involves the dilation of capillaries to increase blood flow;microvascular structural changes and escape of plasma proteins from thebloodstream; and leukocyte transmigration through endothelium andaccumulation at the site of injury. As explained above, the ability tocontrol the inflammatory response is beneficial in reducing inflammationassociated with disease or infection and is also beneficial in treatinga number of diseases with an inflammatory component.

Several types of leukocytes participate in the defense against infectioncaused by foreign invaders (e.g., bacteria and viruses) and tissuedamage due to trauma or inflammation. To fight infection and clear awaydamaged tissue, these cells must move rapidly from the blood stream,where they circulate as unattached, relatively quiescent cells, into theunderlying tissue at sites of infection, inflammation or damage.

Extravasation requires the successive formation and breakage ofcell-cell contacts between leukocytes in the blood and endothelial cellslining the vessels. This successive formation and breakage is also knownas the “leukocyte adhesion cascade”, which is described as a sequence ofadhesion and activation events that ends with extravasation of theleukocyte, whereby the cell exerts its effects on the inflamed tissuesite. Some sources have described the cascade as having at least fivesteps of adhesion termed capture, rolling, slow rolling, firm adhesion,and transmigration. For example, R. O. Hynes and A. Lander, 1992, Cell68:3030, herein incorporated by reference, describes the sequence infive steps. See also, Jung U, Norman K•E, Scharffetter-Kochanek K,Beaudet A L, Ley K, Transit Time of Leukocytes Rolling through venulesControls Cytokine-induced Inflammatory Cell Recruitment In Vivo. Journalof Cinical Investigation. 1998; 102:1526-1533, herein incorporated byreference. Each of these five steps appears to be necessary foreffective leukocyte recruitment, therefore blocking any of the five canseverely reduce leukocyte accumulation in the tissue. These steps arenot phases of inflammation, but represent the sequence of events fromthe perspective of each leukocyte. At any given moment, the events ofcapture, rolling, slow rolling, firm adhesion and transmigration occurin parallel, involving different leukocytes in the same microvessels.

Cells in tissue can adhere directly to one another (cell-cell adhesion)through specialized integral membrane proteins called cell-adhesionmolecules (CAMs). There are four principal classes of CAMs: cadherins(or mucins), the immunoglobulin (Ig) superfamily, selectins, andintegrins. See for example, “Molecular Cell Biology”, 5th Edition,Lodish, et al. Chpt 6, “Integrating Cells into Tissue”, hereinincorporated by reference in its entirety, which describes the generalcategories of CAMs. FIG. 1 is a schematic drawing of these four majorcategories of CAMs. CAMs mediate through their extracellular domainsadhesive interactions between cells of the same type or between cells ofa different type. The homophilic interactions are coordinated by thecadherins and the Ig-superfamily. While the heterophilic interactionsoccur with integrins and selectins.

The structures of the various CAMs vary according to function. Theselectins, shown as dimers in FIG. 1, contain a carbohydrate-bindinglectin domain that recognizes specialized sugar structures onglycoproteins (shown here) and glycolipids on adjacent cells.

Selectins mediate leukocyte-vascular cell interactions. For example,P-selectin is localized to the blood-facing surface of endothelialcells. Selectins contain a Ca²⁺-dependent lectin domain, which islocated at the distal end of the extracellular region of the moleculeand recognizes oligosaccharides in glycoproteins or glycolipids. Forexample, the primary ligand for P- and E-selectins is an oligosaccharidecalled the sialyl Lewis-x antigen, a part of longer oligosaccharidespresent in abundance on leukocyte glycoproteins and glycolipids.P-selectin exposed on the surface of activated endothelial cellsmediates the weak adhesion of passing leukocytes. Because of the forceof the blood flow and the rapid “on” and “off” rates of P-selectinbinding to its ligands, these trapped leukocytes are slowed but notstopped and literally roll along the surface of the endothelium. Amongthe signals that promote activation of the endothelium are chemokines, agroup of small secreted proteins produced by a wide variety of cells,including endothelial cells and leukocytes.

All known selectin ligands are transmembrane glycoproteins which presentoligosaccharide structures to the selectins. Transient bond formationsbetween the selectins and their ligands mediate the early steps of theadhesion cascade. All three selectins can recognize glycoproteins and/orglycolipids containing the tetrasaccharide sialyl-Lewis^(x)(sialyl-CD15). This tetrasaccharide is found on all circulating myeloidcells and is composed of sialic acid, galactose, fucose, andN-acetyl-galactosamine.

PSGL-1 (P-selectin Glycoprotien Ligand) has been characterized as aligand for P-selectin. PSGL-1 is a glycoprotein expressed on blood cellsand contains the sialyl-Lewis^(x) tetrasaccharide. Another P-selectinligand is CD24, which appears to be important for tumor cell binding toP-selectin. For L-selectin, four possible ligands have been identified:GlyCAM-1 (Glycosylation-Dependent Cell Adhesion Molecule), CD34, andMAdCAM-1 (Mucosal Addressin Cell Adhesion Molecule), and PSGL-1.Evidence suggests that both sulfate and sialic acid in an α(2,3) linkageare essential to L-selectin ligand activity. Although specific ligandsfor E-selectin are not yet known, candidate molecules includefucosylated, sialyated oligosaccharides found as components ofglycoprotein and glycolipid molecules.

The P-, E-, and L-selectins have long chains of short consensus repeats,such as SRC, also found in complement regulating proteins. These longchains reach out from the cell surface and at the end of the SRC chainthere is a EGF domain and finally the C-type lectin domain. The S-typeselectins also known as gallectins, bind to gallactose. The collectinshave a collagen like tail, such as mannose binding protein.

Other CAMs include selectin ligands. The sugars that the selectins bindto are mainly sialylated and fucosylated tetrasaccharide antigens calledLewis sugars. Examples include Le^(x) (LPE), 3-sulpho Le^(x) (E) and3-sialo Le^(x) (L), and sulphated tyrosines (of PSGL) (P). The N-linkedsugars are usually bi- or teraantennary. The conformation and linkage ofthe sugars are very important for the overall shape (e.g., a 1-3 and a1-4 linkage can cause the sugars to look completely different).

Integrins are a large family of heterodimeric transmembraneglycoproteins that attach cells to extracellular matrix proteins.Integrins contain large (α) and small (β) subunits of sizes 120-170 kDaand 90-100 kDa, respectively. For tight adhesion to occur betweenactivated endothelial cells and leukocytes, β2-containing integrins onthe surfaces of leukocytes also must be activated by chemokines or otherlocal activation signals such as platelet-activating factor (PAF).Activated integrins on leukocytes then bind to each of two distinctIgCAMs on the surface of endothelial cells: ICAM-2, which is expressedconstitutively, and ICAM-1. ICAM-1, whose synthesis along with that ofE-selectin and P-selectin is induced by activation, does not usuallycontribute substantially to leukocyte endothelial cell adhesionimmediately after activated, but rather participates at later times incases of chronic inflammation. The resulting tight adhesion mediated bythe Ca²⁺ independent integrins-ICAM interactions leads to the cessationof rolling and to the spreading of leukocytes on the surface of theendothelium. The adhered cells then move between adjacent endothelialcells and into the underlying tissue. Integrins contain binding sitesfor divalent cations Mg²⁺ and Ca²⁺, which are necessary for theiradhesive function. Mammalian integrins form several subfamilies sharingcommon β subunits that associate with different α subunits. Integrinligands include NCAM and Fibronectin. These ligands have Ig domains andcan be anchored to membrane by GPI anchor or TM helix. Other CAMsinclude clustered determinants, known as the CD molecules, which mostlyhave Ig domains. Other CAMs include pMHC and TCR; proteoglycans, havinglong unbranched glycans; and growth factors (FGF, EGF and NGF), whichbind to haparan sulphate to be recognized by receptor.

The activated lymphocyte has many glycoproteins and cell adhesionmolecules on its surface, eg TCR, CD43 (hyperglycosylated spacer) andICAM. These molecules are important for the recognition of foreignantigens, activation of APCs and toxic effects on other cells. Thelymphocytes interact with the APC via specific interactions (TCR) andnon-specific interactions (CD2—LFA3 and ICAM-1—LFA1).

Upon infection of tissue, cytokines are secreted that make theendothelial cells express E- and P-selectins on their surface. Thesewill interact with glycoproteins on the surface on the leukocytes in theblood stream, making the cells slow down by a rolling movement. Thetarget of P-selectin is PSGL on the surface of the leukocyte, having aheavily O- and N-linked glycoprotein tyrosine-sulphate content. Thetarget of E-selectin is unknown. The leukocyte also has a selectin onits surface (L). The targets of this are CD34, MAdCAM-1 (both with IgSFdomains and lots of N- and O-linked glycosylation) and GlyCAM-1 (solubleinhibitor of the mucin family). When the leukocyte is stopped it willexrtavasate aided by chemoattractants (such as RANTES) and integrinadhesion to fibronectin of epithelia.

Lymphocytes are capable of a remarkable level of recirculation,continuously moving through the blood and lymph to various lymphoidorgans. About 40-50% of the lymphocytes will be recruited to the spleen,about 38-48% will be recruited to various peripheral lymph nodes and theremainder will be recruited to various mucosal-associated lymphoidtissues (MALT) or SALT (skin associated lymphoid tissues). In order forrecirculating lymphocytes to enter various lymphoid organs orinflammatory-tissue spaces, the lymphocytes must adhere to a passbetween the endothelial cells lining the walls of blood vessels byextravasation. Vascular endothelial cells possess cell adhesionmolecules on their surface. Some of these are expressed constitutivelyand others are inducible (expressed primarily in response to certaincytokines produced during an inflammatory response).

Selectins are membrane glycoproteins with a distal lectin-like domainwhich enables the protein to bind to specific carbohydrate moieties.L-selectin is expressed on all leukocytes while E- and P-selectin areexpressed on vascular endothelial cells. Cadherins (or mucins) areserine- and threonine-rich proteins that are heavily glycosylated. Theypresent sialyated carbohydrate ligands to selectins. For instance, Lselectin on leukocytes recognizes sialylated carbohydrates on twomucin-like molecules (CD34 and GlyCAM-1) expressed on certainendothelial cells of lymph nodes. PSGL-1 on neutrophils interacts with Eand P-selectin on inflamed endothelium. Other CAMs include the categoryknown as Integrins, which generally have a αβ-heterodimers structure.Integrins are integral membrane proteins expressed by leukocytes whichfacilitate adherence to the vascular endothelium. Different integrins,including LFA-1, are expressed by different populations of leukocytes,allowing these cells to bind to different CAMs belonging to the Igfamily expressed along the vascular endothelium. LAD (leukocyte adhesiondeficiency) is an autosomal recessive disease characterized by recurrentbacterial infections and impaired wound healing. Abnormal synthesis ofthe B chain in integrins characterize the LAD disorder. As a result ofthe abnormal synthesis, leukocytes cannot extravasate. The Igsuperfamily of CAMs are proteins which contain at least one Ig-domainsuch as ICAM-1,2,3 and VCAM on vascular endothelial cells. The proteinMAdCAM has both Ig and mucine-like domains expressed by mucosalendothelial cells. It directs lymphocytes into the mucosa. It binds tointegrins via its immunoglobulin-like domain and to selectins via itsmucin-like domain.

During an inflammatory response cytokines and other chemicals act onlocal blood vessels (dilation and increased permeability) to show anincreased expression of CAMs. When this happens the vascular endotheliumis said to be inflamed or activated. Neutrophils are among the firstcells to bind and extravasate. This process is similar for monocytes butbetter studied for neutrophils. Extrasvastion of neutrophils includes:rolling chemoattractant activating stimulus arrest and adhesion andtransendothelial migration rolling. Initially low affinityselectin-carbohydrate interaction is observed during inflammation. E andP selectins expressed in higher levels on vascular endothelial cellsbind to mucin-like cell adhesion molecules on the neutrophil membrane.As the neutrophil rolls, it is activated by various chemoattractantsthat are localized on the endothelial cell surface or secreted locallyby cells involved in the inflammatory process. Chemoattractive cytokines(or chemokines) include IL-8, macrophage inflammatory protein (MIP),PAF, and C5a. Adhesion binding to chemokines triggers G proteinactivated signal transduction pathway, which induces a conformationalchange in integrins, increasing their affinity for Ig superfamily CAMson the endothelium. Extravasation directed migration of the neutrophilthrough interendothelial junctions has not been well characterized.

Antigen specific T cells are known to disappear from the circulation inless than 48 hours. Recruitment of T cells occurs especially atlocations called High Endothelial Venules (HEVs), which are plump,cuboidal cells present in all secondary lymphoid sites except thespleen. Appearance or expression of HEVs dependent upon antigenstimulation. These HEVs express on their surface special CAMs. Antigenstimulation results in an increased expression of these CAMs therebyfacilitating extravasation. Germ-free animals do not have HEVs.Re-circulating lymphocytes, monocytes, and granulocytes bearadhesion-molecule receptors for: CAMs of the selectin family, the mucinfamily (GlyCAM-1 and CD34), and the Ig superfamily (ICAM-1-3, VCAM-1,and MAdCAM-1). When distributed in a tissue-specific way these moleculesare termed: vascular adressins. The corresponding receptors onendothelial cells have come to be called homing receptors. The homingreceptors recruit different subsets of lymphocytes, which migratedifferentially into different tissues based on the expression of thehoming receptors and also due to chemokine secretion patterns anddifferential responses. For example, MIP (macrophage inhibitory protein)preferentially attracts naive T cells. Monocyte chemoattractant protein(MCP) and RANTES preferentially attract memory T cells. Naivelymphocytes initial attachment to HEVs are generally mediated by bindingof homing receptor L-selectin to vascular adressins such as GlycCAM-1and CD34 on HEVs. If the lymphocytes encounter antigens within the node,they become activated and enlarge into lymphoblasts and are retained forat least 48 hours. The lymphoblasts experience rapid proliferation anddifferentiation to effector and memory cells which then leave the node.Effector and Memory Lymphocytes tend to home to regions of infection.Memory lymphocytes are attracted to the type of tissue where theyoriginally encountered the antigen. These cells express high levels ofcertain homing receptors, this allows tissue-specific homing behavior.

Table 1 below summarizes the circulating cellular and endothelialinteractions of several embodiments of the present invention.

The leukocyte cascade sequence is illustrated at FIG. 2. In the absenceof inflammation or infection, leukocytes and endothelial cells liningblood vessels are in a resting state. Inflammatory signals released inareas of inflammation or infection activate resting endothelial cells tomove vesicle-sequestered selectins to the cell surface. The exposedselectins mediate loose binding of leukocytes by interacting withcarbohydrate ligands on leukocytes. Activation of the endothelium alsocauses synthesis of platelet-activating

Endothelial component Cell component Glycosylation L-Selectin (cd62L)Counter receptors on leukocytes Counter receptors O-linked PSGL-1(cd162) glycosylation MadCAM-1 CD34 GlyCAM-1 P Selectin PSGL-1 Counterreceptors O-linked CD-24 glycosylation E Selectin PSGL-1 (hypothesized)Counter receptors O-linked ESL-1 (hypothesized) glycosylation BetaIntegrins LFA-1, MAC-1 Beta integrins glycosylated ICAM-1,2(Intracellular) PECAM-1 (Platelet, endoth) VCAM-1 (Vascular) Neurothelin(cd147) Neurothelin glycosylated CD151 (Transmembrane 4 Glycosylatedsuperfamily protein: PETA- 3, SFA-1) BST-1 (Bone marrow Glycosylatedstromal cell antigen-1) CD18, 29, 49 VLA-4 LPAM-2 I-selectin CD34:FLT-3, FLT-3Ligand CD34 - GlycosylatedFactor (PAF) and ICAM-1, both expressed on the cell surface. PAF andother usually secreted activators, including chemokines, then inducechanges in the shapes of the leukocytes and activation of leukocyteintegrins such as αβ2, which is expressed by T lymphocytes. Thesubsequent tight binding between activated integrins on leukocytes andCAMs on the endothelium (e.g., ICAM-2 and ICAM-1) results in firmadhesion and subsequent movement (extravasation) into the underlyingtissue.

The selective adhesion of leukocytes to the endothelium near sites ofinfection or inflammation thus depends on the sequential appearance andactivation of several different CAMs on the surfaces of interactingcells. It has been found that several CAMs and associated circulatingleukocytes express carbohydrate chains comprising chondroitin sulfateproteoglycans (CSPG).

CSPGs are a family of proteoglycans composed of a core protein andcovalently linked sulfated glycosaminoglycans. Each proteoglycan isdetermined by the glycosaminoglycan side chains. For CSPGs these sidechains are made up of approximately 40 to 100 sulfated disaccharidescomposed of chondroitin 4, 6 and dermatan sulfates. The proteincomponent of the CSPG is ribosomally synthesized and the glycosylationoccurs in the endoplasmic reticulum and Golgi apparatus. The sugarchains are then sulfated at the 4 or 6 positions by severalglycosaminoglycan sulfotransferases.

Disruption of the glycosylation interaction between CAMs and associatedreceptors in the extravasation cascade through targeting of carbohydratechains. A disruption of cell-cell adhesion thus disrupts extravasationof leukocytes into tissue cells. It has been found that the use ofenzymes directed against the CSPG carbohydrate chains allows forregulation and modification of extravasation processes. The enzyme maybe directed against the CSPG carbohydrate chains of ligands expressed oncirculating leukocytes. Thus, the leukocytes are not able to adhere tothe integrins, mucins, or selectins localized on the blood-facingsurface of endothelial cells. As described herein, the ligands forendothelial cell surface CAMs include the ligands for adhesion to theselectin family, the mucin family and the integrin family. Any ligandexpressed on leukocytes containing CSPG carbohydrate chains may betargeted in the several treatments and methods of the present invention.

As used herein, a compound directed against CSPG is a molecule that candegrade CSPG chains, cleave CSPG, bind to CSPG, or inhibit othercompounds or cells from adhering to CSPG. A non-limiting example ofcompounds directed against CSPG includes the chondroitinase enzymes. Theadministration of compounds directed against CSPG may be used to controlextravasation of leukocytes, thus modification of the immune response ispossible.

Thus the use of enzymes directed against CSPG may be used to prevent,treat and alleviate symptoms of inflammation and inflammatory states.The enzyme may also prevent, treat and alleviate symptoms of chronicinflammatory diseases and central nervous system disorders. The enzymemay be used to treat inflammation associated with pain, injection anddiseased states. The enzyme may be used to prevent tissue damage that isassociated with inflammatory processes. Several conditions may benefitfrom controlled immune response. The many types of allergies and many ofthe autoimmune diseases are examples of inflammation in response to whatshould have been a harmless, or at least noninfectious, agent. Someexamples of chronic inflammatory diseases include Asthma, RheumatoidArthritis (RA), Multiple Sclerosis (MS), Systemic Lupus Erythematosus(SLE), and Chronic Obstructive Pulmonary Disease (COPD). The use ofenzymes directed against CSPG may also be used to regulate theinflammatory state associated with one or more disease selected from thegroup consisting of central nervous system disorders, central nervoussystem diseases, spinal cord injury, and cardiovascular diseases.

Inflammatory diseases, autoimmune diseases, and diseases with aninflammatory component may also include Multiple Sclerosis, Meningitis,Encephalitis, Rheumatoid arthritis, Osteo arthritis, Lupus, Wegener'sgranulomatosis, Inflammatory bowel disease: Crohn's colitis, ulcerativecolitis, Asthma, Chlamydia infections, Syphilis, Thyroiditis, Temporalarteritis, Polymyalgia rheumatica, Ankylosing spondylitis, Psoriasis,Vasculitiditis such as: temporal arteritis, Takayasu arteritis,syphilitic aortitis, infectious aneurisms, atherosclerotic aneurisms,inflammatory abdominal aortic aneurysms, polyarteritis nodosa, Kawasakidisease, Churg-Strauss, hypersensitivity vasculitis, Buerger's disease,mesenteric inflammatory veno-occlusive disease, phlebitis,thrombophlebitis, Churg-Strauss, primary angiitis of the CNS, druginduced vasculitis, any secondary arteritis or venulitis, Gout,Pseudogout, Sarcoidosis, Sjogren's Syndrome, Myelitis, Salpingitis ofany etiology, Uveitis, Pelvic Inflammatory Disease, Glomerulonephritisof any etiology, Goodpasture's syndrome, Pericarditis, Myocarditis,Endocarditis, and Pancreatitis.

Suitable enzymes that are directed against CSPGs include chondroitinaseABC type I, chondroitinase ABC Type II, chondroitinase AC andChondroitinase B or mammalian enzymes with chondroitinase-like activitysuch as hyaluronidase 1, hyaluronidase 2, hyaluronidase 3, hyaluronidase4, cathepsins, and ADAMTs, fragments thereof or mixtures thereof.

Chondroitinases are enzymes of bacterial origin that act on chondroitinsulfate, a component of the proteoglycans that are expressed in variousCAMs and receptors associated with inflammatory response. Examples ofchondroitinase enzymes are chondroitinase ABC, which may be produced bythe bacterium Proteus vulgaris (P. vulgaris), and chondroitinase AC,which may be produced by A. aurescens. Chondroitinases ABC and ACfunction by degrading polysaccharide side chains inprotein-polysaccharide complexes, without degrading the protein core.

It has now been found that the CSPG-degrading enzymes such aschondroitinase ABCTypeI, chondroitinase ABCTypeII, chondroitinase AC,chondroitinase B or mammalian enzymes with chondroitinase-like activitysuch as Hyal 1, Hyal 2, Hyal 3, and Hyal 4 are useful in controllingand/or inhibiting the effects of chondroitin sulfates and in developingtherapeutics for the regulation, treatment and prevention ofinflammation associated with various disease states.

Yarnagata et al. (J. Biol. Chem. 243:1523-1535, 1968) describe thepurification of the chondroitinase ABC from extracts of P. vulgaris.This enzyme selectively degrades the glycosaminoglycanschondroitin-4-sulfate, dermatan sulfate, and chondroitin-6-sulfate (alsoreferred to respectively as chondroitin sulfates A, B, and C which areside chains of proteoglycans) at pH 8 at higher rates than it degradeschondroitin or hyaluronic acid. P. vulgaris chondroitinase I migrateswith an apparent molecular mass of about 110 kDa when resolved bySDS-PAGE. The products of the degradation are high molecular weightunsaturated oligosaccharides and an unsaturated disaccharide. However,chondroitinase ABC does not act on keratosulfate, heparin or heparitinsulfate.

Another chondroitinase, chondroitinase II, has also been isolated andpurified from P. vulgaris, Chondroitinase II is a polypeptide of 990amino acids with an apparent molecular mass by SDS-PAGE of about 112kDa. Its molecular mass as determined by electrospray and laserdesorption mass spectrometry is 111,772.+−0.27 and 111,725.+−0.20daltons, respectively. Chondroitinase II has an isoelectric point of8.4-8.45. Its enzymatic activity is distinct from, but complementary to,that of chondroitinase I. Chondroitinase I endolytically cleavesproteoglycans to produce end-product disaccharides, as well as at leasttwo other products which are thought to be tetrasaccharides,Chondroitinase II digests at least one of these tetrasaccharide productsof chondroitinase I digestion of proteoglycan.

Chondroitinase AC and chondroitinase B are chondroitin lyase enzymes,which may be derived from various sources. Any chondroitinase AC or Bmay be used in the present method embodiments including, but not limitedto chondroitinase AC (derived from Flavobacterium heparinum; T.Yamagata, H. Saito, O. Habuchi, S. Suzuki, J. Biol. Chem., 243, 1523(1968)); chondroitinase AC II (derived for Arthobacter aurescens; K.Hiyama, S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama, S. Okada,J. Biochem. (Tokyo), 80, 1201 (1976)); chondroitinase AC III (derivedfrom Flavobacterium sp. Hp102; H. Miyazono, H. Kikuchi, K. Yoshida, K.Morikawa, K. Tokuyasu, Seikagaku, 61, 1023 (1989)); chondroitinase B(derived from Flavobacterium heparinum; Y. M. Michelaaci, C. P.Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), V. M.Michelaaci, C. P. Dietrich, Biochem. J., 151, 121 (1975), K. Maeyama, A.Tawada, A. Ueno, K. Yoshida, Seikagaku, 57, 1189 (1985)); andchondroitinase B (derived from Flavobacterium sp. Hp102; H. Miyazono, H.Kikuchi, K. Yoshida, K. Morikawa, K. Tokuyasu, Seikagaku, 61, 1023(1989)). Suitable chondroitinase AC and chondroitinase B arecommercially available from Seikagaku America, Falmouth, Mass., USA.Additionally, the enzymes may be produced by the methods disclosed inU.S. Pat. No. 6,093,563 by Bennett et al., the disclosure of which isincorporated herein. Chondroitinase I and chondroitinase II are exo- andendo-lyases respectively which cleave both chondroitin and dermatansulfates (Hamei et al 1997). Mammalian enzymes with chondroitinase-likeactivity have been identified. For example, certain hyaluronidases suchas Hyal 1, Hyal 2, Hyal 3, and Hyal 4 also degrade CSPGs and can be usedin the present invention.

Hyaluronidase is an enzyme that catalyzes the breakdown of hyaluronicacid in the body, thereby increasing tissue permeability to fluids. Itis also known as a spreading factor. The hyaluronidase(endo-N-acetylhexosaminidase) enzyme acts on not only hyaluronan butalso chondroitin, chondroitin 4-sulfate and chondroitin 6-sulfate.Besides the hydrolytic reaction, hyaluronidase is also known to catalyzethe reverse reaction, transglycosylation.

Hyaluronidase may be purified from mammalian testicular and spleentissue (common sources are bovine and ovine testes), venom from bees andsnakes, and many bacteria. The enzyme has a molecular weight of 55,000daltons. The function of this enzyme is to catalyze the depolymerizationof mucopolysaccharides, hyaluronic acid, and the chondroitin sulfates Aand C. This enzyme is commonly used in medical practice to encourageintradermal drug absorption in patients and in increasing the efficiencyof local anesthetics.

An enzyme that is directed against both hyaluronic acid andchondoritinase sulfate is also suitable in the several methodembodiments of the present invention. Treponema denticola, Treponemavincentii and Treponema socranskii produce an enzyme that hydrolyseshyaluronic acid and chondroitin sulfate. The secreted enzyme isspecifically inhibited by gold sodium thiomalate and anti-bee-venomantibodies. The affinity-purified extracellular enzyme of T. denticolacontains a single molecular species with a molecular mass of 59 kDa.Since it hydrolyses both HA and CS, this enzyme is termed ahyaluronoglucosaminidase (HGase).

Another embodiment of the present invention is a method of treatinginflammation in a patient comprising extracting circulating cells from apatient, subjecting the cells to an enzyme that cleaves the expressedchondroitin sulfate proteoglycan ex vivo to modify the cells, andadministering the modified blood cells into the patient. Therefore, theuse of enzymes directed against CSPG described herein may also bedirected to ex vivo treatments.

Extraction of cells may be accomplished by a variety of methodsincluding, but not limited to, intravenous blood withdrawal,transfusion, dialysis, bypass, organ transplant and other similarmethods that result in removal of cells from the body. Administration ofthe cells may be accomplished by the same methods used to extract thecells, including, but not limited to, intravenous blood withdrawal,transfusion, dialysis, bypass, organ transplant and the like.

A circulating leukocyte with ligands expressed on its surface containingcarbohydrate chains may be extracted from a patient and modified ex vivoby the presently described enzymes. Extraction may be accomplished byblood draw, transfusion, dialysis, bypass, or organ transplant. Asdescribed, the enzymes would modify the carbohydrate chains,specifically any compound directed against CSPG would be suitable tomodify the extracted leukocytes. Once modified, the leukocytes may bereintroduced into a patient's blood stream. Modified leukocytes will beincapable of adhering to endothelial expressed selectins, mucins, andintegrins. Timing of such extraction and reintroduction into thebloodstream may be optimized by observing the inflammatory response andthe appearance of leukocytes in the blood stream, once said cells aresignaled to specific sites of injury or infection. As a result,extravasation of leukocytes into tissue may be regulated, prevented,reduced, or controlled. Such regulation may be used in methods andtreatments as directed to control and treat inflammatory response anddiseases with an inflammatory component.

Enzyme activity can be stabilized by the addition of excipients or bylyophilization. Stabilizers include carbohydrates, amino acids, fattyacids, and surfactants and are known to those skilled in the art.Examples include carbohydrate such as sucrose, lactose, mannitol, anddextran, proteins such as albumin and protamine, amino acids such asarginine, glycine, and threonine, surfactants such as TWEEN® andPLURONIC®, salts such as calcium chloride and sodium phosphate, andlipids such as fatty acids, phospholipids, and bile salts. Thestabilizers are generally added to the protein in a ration of 1:10 to4:1, carbohydrate to protein, amino acids to protein, protein stabilizerto protein, and salts to protein; 1:1000 to 1:20, surfactant to protein;and 1:20 to 4:1, lipids to protein. Other stabilizers include highconcentrations of ammonium sulfate, sodium acetate or sodium sulfate,based on comparative studies with heparinase activity. The stabilizingagents, such as the ammonium sulfate or other similar salt, are added tothe enzyme in a ratio of 0.1 to 4.0 mg ammonium sulfate/IU enzyme.

Enzymes may be administered intravenously, through local or systemicinjection. Systemic administration is preferable for greater control ofapplication. Other administrations include subcutaneous, transdermal,transmucosal, and enteric coating formulations. The enzymes, singularlyor in combination, can be mixed with an appropriate pharmaceuticalcarrier prior to administration. Examples of generally usedpharmaceutical carriers and additives are conventional diluents,binders, lubricants, coloring agents, disintegrating agents, bufferagents, isotonizing agents, preservants, anesthetics and the like.Specifically pharmaceutical carrier that may be used are dextran,sucrose, lactose, maltose, xylose, trehalose, mannitol, xylitol,sorbitol, inositol, serum albumin, gelatin, creatinine, polyethyleneglycol, non-ionic surfactants (e.g. polyoxyethylene sorbitan fatty acidesters, polyoxyethylene hardened castor oil, sucrose fatty acid esters,polyoxyethylene polyoxypropylene glycol) and similar compounds.

A pharmaceutical carrier may also be used. Suitable carriers includepolyethylene glycol and/or sucrose, polyoxyethylene sorbitan fatty acidesters, polyethylene sorbitan monooleate, and the like, and combinationsthereof.

The dose will also vary depending on the manner of administration, theparticular symptoms of the patient being treated, the overall health,condition, size, and age of the patient, and the judgment of theprescribing physician. Dosage levels of the therapeutic CSPG enzymes forhuman subjects range between about 0.001 mg per kg and about 100 mg perkg per patient per treatment.

The treatment regimens of the present invention may be carried out by ameans of administering chondroitinase ABC_(TypeI), chondroitinaseABC_(TypeII), chondroitinase AC and chondroitinase B or mammalianenzymes with chondroitinase-like activity such as Hyal1, Hyal2, Hyal3,and Hyal4 in vivo or ex vivo. In vivo administration may includesystemic intravenous administration to the area of inflammation. Themode of administration, the timing of administration and the dosage arecarried out such that the inflammation is prevented or reduced bymodifying extravasation of cells. The treatments of the presentdisclosure deliver an effective amount of chondroitinase ABC_(TypeI),chondroitinase ABC_(TypeII), chondroitinase AC and chondroitinase B ormammalian enzymes with chondroitinase-like activity such as Hyal 1, Hyal2, Hyal 3, and Hyal 4, or fragments or combinations thereof to theinjured site. The term “effective amount” means an amount sufficient todegrade the CSPGs or an amount sufficient to modify extravasation ofcells. The effective amount of chondroitinase can be administered in asingle dose or a plurality of dosages.

Although it is to be understood that the dosage may be administered atany time, in one embodiment, the dosage is administered within hours ofthe signaling of inflammatory response, or as soon as is feasible. Inanother embodiment, the dosage is administered to a mammal in one, twoor a plurality of dosages; such dosages would be dependant on theseverity of the injury and the amount of CSPGs present. Where aplurality of dosages is administered, they may be delivered on a daily,weekly, or bi-weekly basis. The delivery of the dosages may be by meansof catheter or syringe. Alternatively, the treatment can be administeredduring surgery to allow direct application to the site of inflammationor injury. Subject to the judgment of the physician, a typicaltherapeutic treatment includes a series of doses, which are usuallyadministered concurrent with the monitoring of clinical endpoints.

The therapeutic CSPG enzymatic agents may be administered by methodsknown in the art, such as by bolus injection, intravenous delivery,continuous infusion, or sustained release formulation, such as implantsand the like.

Formulations suitable for injection are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17^(th) ed. (1985). Such formulations include antibacterialpreservatives, buffers, solubilizers, antioxidants, and otherpharmaceutical adjuncts. Such formulations must be sterile andnon-pyrongenic, and generally will include purified therapeutic porcineB7-1 agents in conjunction with a pharmaceutically effective carrier,such as saline, buffered (e.g., phosphate buffered saline, Hank'ssolution, Ringer's solution, dextrose/saline, glucose solutions, and thelike. The formulations may contain pharmaceutically acceptable auxiliarysubstances as required, such as, tonicity adjusting agents, wettingagents, bactericidal agents, preservatives, stabilizers, and the like.

EXAMPLES Example 1

Toxicity Studies. Female Long Evans rats from Charles RiverLaboratories, weighing approximately 210 grams were housed in the AcordaAnimal Care Facility for 5 days prior to injection to ensure health andweight stability. Rats were anesthetized with isoflurane and injectedi.v. via tail veins with chondroitinase ABC I (Seikagaku; Cat number100332, lot number E02201). Animals were injected with either 0, 0.2,0.775 or 7.775 mg/kg with solutions containing 0, 0.2, 0.775 and 7.775mg/ml, respectively in Hank's balanced salt solution.

Additionally, toxicity studies were conducted for Intrathecal (IT)catheter administration. Intrathecal catheters were placed in 16 normal,un-injured female rats at about the T13/L1 vertebral junction fordelivery of chondroitinase. Catheters were fed rostrally to rest at theT9/T10 level to simulate previous chondroitinase studies. Twenty-fourhours after intrathecal catheter placement animals were dosed with 0,0.06, 0.6 or 6.0 Units of Acorda chondroitinase ABCI (100Units/milligram) in 20 microliters of artificial cerebrospinal fluidover a 20 minute period.

Animals were observed for 24 hours or 7 days and their weights andtemperatures were followed. Animals were monitored for pain and distressand body weights were acquired daily. No overt reactions were observedduring or immediately after chondroitinase ABCI dosing. No swelling,inflammation, bruising or necrosis was noted at the injection site forthe IV experiment. No changes in body temperature were observed inanimals treated via the IT route. No alterations in feeding, grooming orvocalizations were noted. Animals were assessed for motor behavior in anopen pool. No abnormalities were noted by the animal care staff orbehavioral specialists. Animals displayed no signs of joint tendernessor swelling. There were no significant differences in weight changebetween the treatment groups. The results demonstrate thatchondroitinase ABCI treatment is not associated with acute toxicityusing IV doses. Additionally, Chondroitinase appears safe in singleintrathecal (I.T.) doses.

What has been described and illustrated herein are embodiments of theinvention along with some of their variations. The terms, descriptionsand figures used herein are set forth by way of illustration only andare not meant as limitations. Those skilled in the art will recognizethat many variations are possible within the spirit and scope of theinvention, which is intended to be defined by the following claims andtheir equivalents in which all terms are meant in their broadestreasonable sense unless otherwise indicated.

What is claimed:
 1. A method of treating inflammation in a patientcomprising: extracting cells circulating in the blood stream of apatient; subjecting the cells to an enzyme that cleaves chondroitinsulfate proteoglycan ex vivo to modify the cells, wherein the cells areselected from the group consisting of neutrophils, eosinophils,basophils, lymphocytes, B-cells, T-cells, monocytes, and macrophages;and administering the modified cells to the patient.
 2. The method ofclaim 1, wherein the enzyme is selected from the group consisting ofchondroitinase ABC Type I, chondroitinase ABC Type II, chondroitinaseAC, chondroitinase B, Hyaluronidase 1, Hyaluronidase 2, Hyaluronidase 3,Hyaluronidase 4, and hyaluronoglucosaminidase.
 3. The method of claim 1,wherein the enzyme is directed against integrins containing chondroitinsulfate proteoglycans expressed on the surface of the cells circulatingin the blood stream.
 4. The method of claim 1, wherein the cells areextracted from the blood stream of the patient after activation of theimmune response of the patient.
 5. The method of claim 1, wherein thecells are extracted from the blood stream of a patient prior toactivation of the immune response of the patient, and the modified cellsare administered to the patient after the activation of the immuneresponse.